Secretion factors for gram-positive microorganisms genes encoding them and methods of using it

ABSTRACT

Heterologous protein secretion from Gram-positive bacteria, in particular from Bacilli has, with few exceptions, met with little success. Incompatibility of the heterologous proteins with the protein secretion machinery of the host is the main cause of this effect. This limiting factor for the production of heterologous proteins in commercially significant concentrations from  Bacillus subtilis  is removed by overexpressing the  Bacillus subtilis  protein FtsY or FtsY protein in combination with overexpression of other members of the bacterial signal recognition particle. Said gene(s) is(are) overexpressed in Bacillus host cells expressing a heterologous protein which then shows an increased amount of the heterologous protein secreted in the surrounding medium.

FIELD OF THE INVENTION

This invention relates to processes for the production of proteins bymicro-organisms. Specifically, it relates to the secretion ofheterologous proteins by micro-organisms, in particular by Gram-positivebacteria, especially by the bacterial host Bacillus.

BACKGROUND OF THE INVENTION

It also relates to (the overexpression of) a novel gene encoding aprotein involved in the early stages of prokaryotic protein secretion.specifically, it relates to the overexpression of said gene within aBacillus host (over) expressing heterologous proteins.

B. subtilis and (closely) related bacilli secrete proteins directly intothe growth medium to high concentrations. Secretion as a mode ofproduction of proteins of interest, be it homologous to the host orheterologous to the host, be it of recombinant origin or not, providesseveral advantages over intracellular production. It for instancefacilitates purification of the product, it theoretically will lead to ahigher yield, no aggregation of the product will occur, and it gives thepossibility for continuous cultivation and production. However, attemptsto secrete heterologous proteins from B. subtilis and (closely) relatedorganisms at commercially significant concentrations have, with fewexceptions, met with little success.

Nearly all secreted proteins use an amino-terminal protein extension,known as the signal peptide, which plays a crucial role in the targetingto, and translocation of precursor proteins across the membrane andwhich is proteolytically removed by a signalpeptidase during orimmediately following membrane transfer. The newly synthesized precursorproteins are recognized by specific proteins in the cytoplasmcollectively called chaperones. These chaperones prevent polypeptides,destined for translocation, to aggregate or fold prematurely leading toan export incompatible conformation.

For instance, SecB, GroEL/GroES and DnaK/DnaJ are the presently knownchaperones in the export pathway of E. coli. For the productive bindingof precursor proteins to translocation sites in the cytoplasmic membraneSecA is needed. SecA, a protein of which cytoplasmic, peripheral as wellas integral membrane forms have been detected, has an ATPase activitywhich mediates the initial channelling of precursor proteins into theexport pathway.

The SecA subunit acts as a receptor recognizing the leader and maturedomains of the preproteins (Lill et al. 1990) as well as the SecBchaperone (Hartl et al. 1990). It has been suggested that SecApenetrates into the membrane, after binding of ATP, and so promotes thecoinsertion of the preprotein. After hydrolysis of bound ATP thepreprotein is released from the SecA protein (Schiebel et al. 1991).Translocation is completed with the proton motive force as the maindriving force and requires members of the integral membrane part of thepreprotein translocase complex like SecY, SecE and SecG (p12/Band1).SecD and SecF are also integral membrane proteins and are probablyparticipating in the late steps of protein translocation.

For many years, the protein secretion machinery in prokaryotes has beenconsidered to be independent from the protein secretion system found inhigher eukaryotes (Luirink et al, 1992)a. In mammalians, targeting ofsecretory proteins to the endoplasmic reticulum (ER) is mediated by thesignal recognition particle (SRP), which is a ribonucleoprotein particlecomposed of one RNA molecule SRP 7S RNA) and six polypeptides of 9, 14,19, 54, 68 and 72 kD. The SRP proteins are associated with the RNA aseither monomers (SRP19 and SRP54) or heterodimers (SRP9/14 andSRP68/72).

As soon as the signal peptide of secreted and transmembrane proteins hasemerged from the ribosome, it is recognized and bound by SRP, which alsohas affinity for the ribosome. This association slows down theelongation of the polypeptide chain (elongation arrest). When thecomplex of SRP, nascent polypeptide chain, and ribosome bind to the SRPreceptor (SR or docking protein) associated with the ER membrane, thenascent polypeptide chain is displaced from SRP in a GTP-dependentreaction and protein translation is resumed.

The translocation of the polypeptide into the ER takes placeco-translationally through a protein pore, the translocon (Gilmore etal. 1993). Thus, the SRP functions both as a cytosolic chaperonepreventing premature folding of the preprotein by coupling translationto translocation and as a pilot to guide the preprotein to the SRPreceptor complex in the membrane. The 54 kD subunit of SRP (SRP54) bindsto the signal peptide when it emerges from the ribosome and thereforeseems to have a key function in the SRP-mediated process of proteinsecretion.

Today more and more data become available indicating that anSRP-mediated export pathway may also function in other organisms.Homologues of mammalian SRP components have been isolated from Yeast(Hann et al. 1989), E. coli (Bernstein et al. 1989, and Rymisch et al.1989), Mycoplasma mycoides (Samuelsson, 1992) and Bacillus subtilis(Struck et al. 1989, and Honda et al. 1993).

So it is likely that an SRP-mediated pathway functions in prokaryotes ina separate secretory pathway or may form part of the general secretorypathway.

In E. coli members of an SRP-like secretory pathway were identified.These members are Ffh (Fifty four homologue) and a 4.5S RNA moleculewhich are homologous to the SRP54 and SRP 7S RNA0f eukaryotic SRP (Ribeset al. 1990). It is shown that Ffh interacts specifically with thesignal sequence of nascent presecretory proteins (Luirink et al. 1992).E. coli protein FtsY, which originally has been implicated in celldivision (because its gene is located in an operon together with FtsEand FtsX) displays striking sequence similarity with the subunit ofmammalian docking protein. Several observations suggest that FtsY is thefunctional E. coli homologue of the mammalian SRP receptor (Luirink etal. 1994). Depletion of either FtsY, Ffh or the RNA component of the E.coli SRP affects the export of several secretory proteins.

Also in B. subtilis components of the SRP-like secretory pathway havebeen found. The Small Cytoplasmic RNA (scRNA) was shown to have afunctional relationship with the human SRP 7S RNA and the E. coli 4.5SRNA (Nakamura et al., 1992). The B. subtilis scRNA is transcribed fromthe scr gene as a 354 nucleotide precursor which is then processed to a271 nucleotide RNA at the 5′ and 3′ end (Struck et al., 1989), which issimilar to its eukaryotic homologue (300 nucleotides) but much largerthan the E. coli 4.5S RNA (114 nucleotides). Also the secondarystructure of the scRNA is very similar to the eukaryotic SRP 7S RNA,lacking only the it domain III (Struck and Erdmann, 1990). This is incontrast to the other eubacterial SRP-like RNAs, which only fold into asingle hairpin corresponding to domain IV (Poritz et al., 1988).Therefore the B. subtilis scRNA is both in size and secondary structurean intermediate between prokaryotic and eukaryotic SRP-like RNA.

Besides the =scr gene another gene encoding a SRP constituent has beenisolated from B. subtilis. The ffh gene was found to encode the Ffhprotein which shows homology to both the E. coli and eukaryotic SRP54protein (Honda et al., 1993).

It is not unlikely that chaperones or members of the SRP-like secretionpathway may become a rate-limiting step in the secretion pathway, theresult of which being that the majority of the heterologous proteinexpressed will aggregate or fold prematurely. This effect could be thereason why attempts to secrete heterologous proteins in high amountsfrom Gram-positive micro-organisms, in particular B. subtilis and(closely) related micro-organisms have met with little success.Overexpression of particular members of the B. subtilis secretionmachinery, especially of chaperone-like proteins which are therate-limiting step in the secretion pathway would solve this problem. Itis to be understood that the terms “chaperone” and “secretion factor”are not completely clearly defined. Both groups of proteins will atleast overlap and in some cases may be identical. Because the mechanismof action of these proteins is not yet clearly understood, both termswill be used interchangeably herein.

SUMMARY OF THE INVENTION

The invention thus provides a proteinaceous substance comprising atleast a functional part of a chaperone-like protein expressed byGram-positive bacteria encoded by the ftsY gene of said bacteria, arepresentative of said gene being defined by the seguence of seq. ID no.7.

When Gram-positive bacteria, especially Bacillus species and inparticular Bacillus subtilis and its closely related organisms areprovided with this proteinaceous substance, of which the functionalityis defined as being able to recognize a protein of interest to besecreted and lead it into the secretory pathway, they will have anenhanced capability of secreting proteins. It is very likely thatproteins to be secreted must have a signal sequence, which may be theirown signal sequence, or a signal sequence of a homologous protein of thehost bacteria or a signal sequence homologous to the micro-organism fromwhich the proteinaceous substance, i.e. the secretion factor accordingto the invention is derived.

The protein of interest may be any protein which up until now has beenconsidered for expression in prokaryotes, as long as it can be providedor has of its own a signal sequence which render it suitable forsecretion in a Gram-positive host. Of course it must also be able to berecognized (if possibly not very efficiently) and lead into thesecretory pathway by the chaperone-like proteins according to theinvention. It may not be the case that the chaperone-like proteins wouldbe capable of recognizing and leading into secretion each and everyprotein by itself. Other secretion factors may become the rate-limitingstep, if the presently invented secretion factor is provided insufficient quantities. In that case it is preferred to also provide thehosts with the secretion factors which may become the rate-limiting stepin sufficient quantities also. since we believe that there is a SRP-likeroute in Bacillus species and other gram-positive bacteria, it would beadvantageous to provide the micro-organism with enhanced amounts ofFtsY, the 7S scRNA and Ffh.

The protein of interest may be either homologous or heterologous to thehost. In the first case overexpression should be read as expressionabove normal levels in said host. In the latter case basically anyexpression is of course overexpression.

The proteinaceous substance according to the invention, which forconvenience will often be referred to as the chaperone, secretion factoror the chaperone-like protein, may be homologous to the host, which ispreferred, but it may also be heterologous to the host, as long as it iscompatible with the secretion machinery of the host. It stands to reasonthat this will be most likely in closely related organisms. Thus in thecase of a Bacillus subtilis secretion factor, it would be preferred touse it in a Bacillus.

The sequence being depicted as giving a representative of a sequenceencoding a chaperone-like protein according to the invention is given inorder to enable the person skilled in the art to find homologoussequences which encode similar or functionally the same chaperone-likeproteins in other Gram-positive bacteria, in particular of otherBacillus species. Given the general level of skill in the art, it willbe routine work to prepare for instance primers based on the givensequence and to screen for other homologous sequences encoding saidchaperone-like proteins These chaperone-like proteins from other relatedorganisms should therefore be considered as part of the presentinvention. Their DNA and/or amino acid sequences usually will be quitehomologous. As a rule the homology will be greater then 70% overall, inparticular homologies of greater than 85% overall are to be expected. Itis understood that all homologous genes which can hybridize with thesequence depicted in seq. ID no. 7 and which encode a protein ofessentially the same structure or function are comprised in thisinvention. The following equation, which has been derived from analyzingthe influence of different factors on hybrid stability:

Tm=81+16.6(log 10 Ci)+0.4(% G+C)−600/n−1.5%

mismatch (Ausubel et al., supra) where

n=length of the shortest chain of the probe

Ci=ionic strength (M)

G+C=base composition,

can be used to determine which level of homology can be detected usingDNA-DNA hybridisation techniques.

Therefore the term “essentially of a structure” is intended to embracesequences which can include conservative mutations, where the sequenceencodes the same amino acid, but which may differ by up to 35% in theDNA sequence according to the above equation, more typically by up to10%. It is not always necessary to have a complete chaperone-likeprotein to perform the functions of recognizing the protein to besecreted and leading said protein into the secretion pathway. Wherepossible the hosts may thus also be provided with functional parts ofsaid secretion factor. It is also possible and even likely thatassociation with non-protein material such as the 7S RNA may occur whenthe secretion factor performs its functions. The term proteinaceoussubstance is chosen to include such associations. It is by now wellknown in the art that mutations in proteins may lead to higher activity,longer half-lives, better stability of the mutated protein. Suchderivatives of the secretion factors according to the invention are alsopart thereof, since given the information presented herein, it isroutine work to find weak spots, or other sites interesting for mutationin the secretion factors according to the invention and makingsite-directed mutations.

A preferred embodiment of the present invention is a proteinaceoussubstance which is a chaperone-like protein which is at least partlyencoded by the ftsY gene of a Bacillus species. Bacillus species arehighly preferred organisms to express genes of interest in and a lot ofdevelopmental and production experience is available. As stated beforehowever, there has always been a problem with secretion of especiallyheterologous proteins from Bacillus. This problem may in many cases besolved by providing Bacillus organisms with chaperone-like proteins fromother related species, but it will be clear that chances of a goodfunctional secretion factor in Bacillus, including recognition of theheterologous protein are highest using a chaperone-like protein which isderived from a secretion factor in a Bacillus species. Of specialinterest as a chaperonelike protein is a proteinaceous substance whichis at least partly encoded by the ftsY gene of Bacillus subtilis oranother Bacillus species. These proteinaceous substances are very likelyto be analogues of the eukaryotic docking protein (or SRP receptor) asis the case with products derived from the E. coli tax gene. Lack ofsufficient amounts of this chaperone-like protein will definitely have agreat influence on the capability of host micro-organisms to secrete anyproteins, let alone heterologous proteins. At present we believe thatheterologous protein may be predominantly secreted using the SRP-likeroute, i.e. by binding to Ffh, the 7s RNA and the FtsY, whereashomologous proteins use the general secretion pathway. It is also clearthat if a heterologous protein to be secreted is provided with a signalhomologous or very closely related to a signal present in Bacillussecretory proteins that the presence of a sufficient amount of thesecretion factor which normally has the function of recognizing such asignal will lead to enhanced secretion.

The preferred method of providing a Gram-positive bacteria, inparticular a Bacillus species with the possibility of expressingsufficient amounts of the chaperone-like proteins (and for instancescRNA) according to the invention is of course by providing saidmicro-organism with the genetic information to overexpress saidchaperone-like protein. The invention therefor also provides arecombinant DNA molecule comprising at least a part of an ftsY geneencoding a chaperone-like protein of Gram-positive bacteria, said partencoding at least a functional part of said chaperone-like protein,whereby a representative of said gene has the sequence of seq. ID no. 7.

As is true for the proteinaceous substances of the invention the givensequence is given for the reason of enabling the skilled person to findhomologous sequences encoding similar secretion factors. Variants mayexist within Bacillus species and other Gram-positive bacteria. It willalso enable the person skilled in the art to construe silent mutations,to construe beneficial mutations or mutations having no effect on theactivity of the chaperone resulting from expression. For differentspecies codon preference may de different, degeneracy may be accountedfor. All these modifications should be considered to be within the scopeof the present invention. To define which genes still belong to theinvention can really only be done by their functionality. If they encodea substance which has the same activity (in kind, not in amount) as thepresently invented chaperone-like proteins then the gene (or therecombinant DNA molecule) should be considered to belong to the presentinvention, if the molecule is derived from a Gram-positive bacteria , inparticular a Bacillus species. Usually this will coincide with a ratherhigh degree of homology for instance of 70-95% overall.

A further preferred embodiment of the present invention is of course agene or a recombinant DNA molecule comprising at least a part of theftsY gene of a Bacillus species. The main reason for this preference isof course that Bacillus species are well known production organisms inwhich for reasons already mentioned it would be helpful to provide anautologous (sometimes also called homologous) chaperone-like protein.The most preferred chaperone-like protein at the present time is the oneencoded by a recombinant DNA molecule comprising at least a part of theftsY gene of Bacillus subtilis.

For easy transfer of the genetic information of the secretion factorsaccording to the invention it is preferred to provide the recombinantDNA molecule as a vector. The invention thus also provides a recombinantvector comprising a recombinant DNA molecule as disclosed above andsuitable regulatory elements for replication and/or expression. Thenature and kind of such a vector is not important, as long as it iscapable of transferring the wanted genetic information into the desiredmicro-organism and preferably being capable of replicating or beingreplicated in such micro-organism. They may comprise many additionaladvantageous features such as marker genes, restriction sites, etc.Chromosomal integration of (part of) the gene according to the secretionfactor is also comprised within this invention. It would of course beadvantageous to only have to transfer a micro-organism with one vector.Preferably the invention provides a recombinant vector as describedabove further comprising a gene encoding a protein of interest to besecreted.

The invention further provides micro-organisms which have been providedwith the genetic information to encode a chaperone-like according to theinvention by whatever method. The invention thus includes a cell derivedfrom a Gram-positive host cell comprising a recombinant DNA molecule ora vector as defined herein before. Preferably the cell is derived from aBacillus species. In a further preferred embodiment the cell has alsobeen provided with the ability to overexpress either or both 7S scRNAand Ffh in a similar manner as it has been provided with the(over)expression of FtsY.

In a further preferred embodiment the cell also has been provided withthe ability to overexpress a homologous protein or to express aheterologous protein. A suitable way to arrive at such a cell isproviding it with the genetic information for said protein of interest,leading to a cell comprising a vector having the genetic informationencoding a chaperone-like protein according to the invention, furthercomprising a vector comprising a gene encoding a protein of interest tobe secreted. All methods leading to the products of the invention are ofcourse also part of this invention. In particular important are themethods leading to the enhanced production of proteins secreted in theculture medium. The invention thus also includes a method for enhancingthe secretion of a protein of interest from a Gram-positivemicro-organism, comprising the steps of providing said micro-organismwith the possibility to over express the protein of interest, providingthe micro-organism with the possibility of overexpressing aproteinaceous substance according to the invention, and culturing saidmicro-organism under suitable conditions.

Preferably the possibility to overexpress the protein of interest isprovided by a vector as disclosed herein before and the possibility tooverexpress a proteinaceous substance according to the invention is alsoprovided by a vector as disclosed hereinabove.

The invention will now be further illustrated in the following detaileddescription and the examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. FIG. 1A shows the plasmids pHB201 and pHBNde described inExample 1. FIG. 1B shows the plasmid pHBChap.

FIGS. 2A (SEQ ID NOS:9-13) and 2B (SEQ ID NOS:14-19) illustrate sequencehomology among various ORFs as described in Example 2.

FIG. 3 shows molecule features the DNA sequence of the Pst I fragmentdescribed in Example 3 (SEQ ID NO:20)

FIG. 4 illustrates the GTP binding boxes of SRα like proteins asdescribed in Example 3.

FIG. 5 shows the chromosomal organization of the genes in the Pst Ifragment described in Example 3.

FIG. 6 shows the integration construct pNS′ as described in Example 4.

FIG. 7 illustrates the double cross-over event as described in Example5.

FIG. 8 illustrates the chromosomal organization of an isolated integrantstrain as described in Example 5.

FIG. 9 shows an SDS PAGE of h-IL-3 as described in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

There is now growing evidence that poor expression and/or secretion iscaused by incorrect folding of the heterologous protein in the hostcell. The cause of this effect may be the incompatibility of the hostcell's chaperone-like proteins of the regular secretory pathway and theheterologous protein. As a result the newly synthesized heterologousproteins will be recognized very inefficiently and in this way become arate-limiting step in the translocation process. This will be even morepronounced if the heterologous protein is overexpressed. One possibilityto overcome this problem is to express heterologous chaperone-likeproteins which are homologous to the heterologous protein which is to besecreted. Expression of E. coli SecB in B. subtilis has shown tofacilitate secretion of the SecB-dependent maltose-binding protein of E.coli (Collier, 1994). This option is probable not applicable when theheterologous protein and secretion factor are from a more phylogeneticdistant organism. In this way the host cell's regular secretionmachinery could become incompatible with the heterologous chaperone-likeprotein itself, leaving the same effect: extreme low secretionefficiency. Another possibility, part of this invention, is tooverexpress one or more of thee host cell's chaperone-like proteins,preferably the SRP-like chaperone-like proteins and so increase theavailability of these chaperone-like proteins for the heterologousprotein.

Because homologues of SecA (Sadaie et al. 1991), SecE (Jeong et al.1993), SecY (Su et al. 1990), and Lep (Van Dijl et al. 1992) have beenidentified in B. subtilis, it is suggested that signal peptide-dependentprotein secretion in B. subtilis utilizes a Sec-pathway that is similarto that of E. coli. So far SecB, which is considered to be the majorchaperone in E. coli, seems to be the only chaperone which has a directbinding affinity for SecA and so contributes to the accurate targetingof the preprotein-SecB complex to the membrane bound translocase. TheSecB protein is needed for only a subset of the envelope proteins soSecB independent proteins will enter the Sec-pathway with the aid ofhelper proteins like GroEL/GroES, DnaK/DnaJ or other proteins like SRP.In eukaryotic organism SRP mainly is responsible for the translocationacross the ER membrane. Recently more evidence has become available ofthe existence of an SRP mediated secretion route in bacteria. Becausethe eukaryotic pathway has probably evolved from the bacteria it isthinkable that said proteins are also dependent on this pathway whensaid proteins are expressed in bacteria like Bacillus. Thus optimisationof this particularly pathway in Bacillus will be more profitable forheterologous (eukaryotic) proteins secretion than the optimisation ofthe well known sec-pathway. This invention relates to the cloning of theBacillus ftsY gene and its effect after (over)-expression, alone or incombination with other members of the bacterial SRP, upon heterologousproteins.

For the cloning of B. subtillis ftsY degenerate primers were synthesizedmaking use of the existing homology boxes between the SRα homologues ofdifferent organism (FIG. 2a). After an inversed PCR reaction using a 110bp fragment, which was derived from a nested PCR reaction, as template a4 kb fragment could be detected. Sequencing results (FIG. 2b) revealedan open reading frame of 329 amino acids. This protein shared 48.2%amino acid identity and 65% similarity with the ftsY gene of E. coli.

As will be shown in the Examples, the hybridizing experiments originallylead to an unwanted result, i.e. a smear of indistinct bands.surprisingly, we were able by cutting out a region around the expectedsize of the amplified fragment and applying PCR to that region again inresolving this smear into a group of distinct bands. Unfortunatelyhardly any band was seen at the expected size of the fragment thatshould have been amplified. However, in a third round of amplificationwe were nevertheless able to obtain a fragment which could be usedfurther.

EXAMPLE 1 Construction of a Promoter Vector for Secretion factorOverexpression

pHB201 is capable of autonomous replication in both E. coli (high-copy)and Bacillus (low-copy) strains. This plasmid confers resistance to theantibiotics chloramphenicol and erythromycin in E. coli and Bacillus.Further the plasmid carries a CAT86::lacZ fusion gene preceded by thestrong Lactococcus lactis promoter 59 which also act as strong promoterin B. subtilis (Van der Vossen et al. 1987).

By replacing the SalI/EcoRI fragment of this plasmid by a synthetic DNAfragment (SEQ ID NO:1) the CAT86::lacZ was deleted and an unique NdeIrestriction site introduced overlapping the translation initiation sitegenerating pHBNde (FIG. 1a). This allows us to express the chaperonegenes directly downstream the strong Lactococcal promoter 59 withoutcreating fusion proteins as would be with the original pHB201 vector(FIG. 1b).

EXAMPLE 2 Molecular Cloning of Bacillus subtilis DNA FragmentsHomologous With the Human SRα Gene

A set of three Polymerase Chain Reactions (PCR) were performed asfollows. Chromosomal DNA of B. subtilis 168 was used as template in afirst PCR reaction with degenerate primers AB4229 (SEQ ID NO: 2) andAB4230 (SEQ ID NO: 3). After 30 cycles and an annealing temperature of40° C. the amplified DNA was fractionised by electrophoresis on a 2%Metaphor (FMC BioProducts) agarose gel. The results showed a smear ofill resoluted bands. DNA fragments ranging in size from 220 bp to 300 bpwere purified from the agarose gel with a QIAquick gel extraction column(QIAGEN).

1/50 of these isolated fragments were used in a second PCR withdegenerate primers AB4229 and AB 4241 (SEQ ID NO: 4) using the samereaction conditions as in the first PCR. The result of this PCR showed anumber of district bands, however, a band of the expected size (±120 bp)was hardly visible. Fragments of ±120 bp were isolated from the agarosegel as above and used in a third PCR with the same primers andconditions as were used for the second PCR. The resulting singlefragment was isolated, purified and after treatment with T4polynucleotide kinase ligated into dephosphorylated pUC18 linearizedwith SmaI.

After electroporation to E. coli JM109, selection on IPTG/X-gal plates,the DNA from six white colonies were used for automated sequencing.Within all six isolates an Open Reading Frame (ORF) could be detected.This ORF showed over 70% similarity with an alignment of homologs fromseveral other organisms (FIG. 2b).

EXAMPLE 3 Sequencing of Unknown DNA Sequences Adjacent to a ShortStretch of Known Sequence

By using a labelled internal ftsY fragment derived from example 2 wecould detect a single 4 kb PstI band in a hybridisation experiment. Noneof the attempts to clone this fragment directly into pUC weresuccessful, indicating that cloning of this fragment could be lethal inE. coli. An inversed PCR (IPCR) was used for determination of thesequence.

A total PstI digest of B. subtilis 168 chromosomal DNA was used astemplate in the IPCR with primers AB5356 (SEQ ID NO: 5) and AB5357 (SEQID NO: 6). The resulting fragment was used directly for automatedsequencing making use of the same primers. The sequence of the rest ofthe PstI chromosomal DNA fragment located upstream of the primer AB5357and downstream of primer AB5357 was determined by automated sequencingmaking use of newly developed primers while the sequence was unveiled.The total 4370 bp DNA sequence of the PstI fragment is shown in FIG. 3.

Analysis of the sequence showed the presence of several Open ReadingFrames (ORFs), including the one for FtsY. When comparing the overallstructure of the SRα like proteins and SRP54-like proteins a commondomain is evident which comprises GTP binding boxes (the G-domain, seeFIG. 4). Also from this figure it is clear that the B. subtilis FtsYprotein contains only a very short N-terminal domain, in contrast to theeukaryotic and E. coli homologues. Since the N-terminal domain in thoseorganisms serves as a membrane anchor it is possible that in B. subtilisFtsY functions with a different mechanism, and is possibly morechaperone-like in its action, although it may still be membrane-bound.

Analysis of the sequence showed the presence of several more ORF's,including a truncated ORF showing homology to the Ffh protein, and atruncated ORF showing homology to several DNA segregation proteins likethe Yeast protein SMC1.

The chromosomal organisation of the genes in the PstI fragment is shownin FIG. 5.

EXAMPLE 4 Effect of FtsY Depletion Upon the Secretion of HeterologousProteins

The effects of depletion of FtsY in Bacillus on the processing and/orsecretion of heterologous proteins were studied by placing thechromosomal ftsY gene under control of the inducible SPAC promoter(Yansura et. al). For this the N-terminal part of the ftsY gene wascloned into the multiple cloning site of pDG148 directly downstream theSPAC promoter. The SPAC-ftsY-penP-lacI fragment from the resultingpDGFtsY′ plasmid was recloned into pPPNeo2 making the final integrationconstruct pNSFtsY′ (FIG. 6).

pNSFtsY′ is capable of autonomous replication in E. coli but not inBacillus. It confers resistance to the antibiotic ampicillin which canbe used for selection in E. coli and neomycin for selection in Bacillus.Integration of pNSFtsY′ into the ftsY locus results in a truncated copyof ftsY (ftsY′) under control of the authentic promoter and an intactcopy of ftsy under control of the SPAC promoter.

Neomycin resistant pNSFtsY′ integrants could be selected after aprotoplast transformation of B. subtilis 168. Integrants growing in amedium with 0.5 mM IPTG showed growth characteristics comparable to B.subtilis host lacking the integrated plasmid. The growth rate ofintegrants did not decrease after incubation in the absence of IPTG, nordid the cell morphology.

Effects of depletion of FtsY on protein translocation were examined byfermentation experiments using hosts expressing heterologous proteins. ApUB110 like vector containing regulatory sequences of the B.licheniformis or B. amyloliquefaciens α-amylase gene was used for theexpression of heterologous proteins. Some of the heterologous proteinswere produced to slightly higher levels in cells cultured in thepresence of IPTG, however in the absence of IPTG the production of theheterologous proteins was not completely abolished.

These effects were unexpected since in E. coli depletion of FtsY has aprofound effect on the cell morphology and growth rate. Therefore it ispossible that there is FtsY formation despite the absence of IPTG,indicating transcriptional read through. It should be possible tocorrect this by insertion of a strong terminator signal upstream of theSPAC promoter in the integration construct. To confirm that the absenceof FtsY is hazardous to the cell attempts were made to disrupt the ftsYgene.

EXAMPLE 5 Construction of Bacillus Strains Without a Functional ftsYGene

Since the experiments described in Example 4 were unexpected in thesense that strain containing the ftsY gene under the control of the SPACpromoter were still viable, and showed no clear phenotype in the absenceof the inducer IPTG, we hypothesized that the construct we used wasleaky so that even in the absence of IPTG a small amount of FtsY wouldbe produced. To eliminate the production of FtsY we tried to disrupt theftsY gene by insertion of a neomycin resistance marker. We constructed aplasmid pBHdsPtsYNeo which harbours the 5′ and the 3′ end of the ftsYgene separated by the Neomycin resistance gene in a vector unable toreplicate in B. subtilis.

Plasmid pBHdSFtsYNeo was linearized by cutting with Hpal and used totransform B. subtills 168 to neomycin resistance. This should lead tostrains having the original ftsY gene replaced by the fstY::Neoconstruct via a double cross-over event (see FIG. 7). However, we wereunable to select for Neomycin resistant colonies using this linear DNA,suggesting the possibility that disruption of the ftsY gene is lethal tothe cell.

We therefore repeated the transformation experiments with intact, uncut,plasmid DNA. In this case integration of the plasmid into the ftsY genecan take place via a single cross-over event (Campbell typeintegration), leading to neomycin resistant colonies which have both anintact and a disrupted copy of the ftsY gene present (see FIG. 8). Weisolated integrant strains, one of which was shown to have thechromosomal organization represented in FIG. 8. Since in this strain twocopies of the 3′ end of the fstY gene are present recombination betweenthem is possible, leading to excision of the plasmid sequences inbetween and formation of a strain containing only one copy of the ftsYgene, disrupted by the neomycin resistance marker. Despite severalattempts we were unable to isolate such recombinant strains, suggestingagain that such strains are unviable.

EXAMPLE 6 Effect of FtsY Overexpression on the Location of Precursorsand Mature Heterologous Proteins

The effects of overexpression of FtsY in Bacillus on the processingand/or secretion of heterologous proteins were studied by placing thecomplete ftsY gene under control of the constitutive P59 promoter inpHBNde (see Example 1) resulting in the plasmid pHBFtsY. Effects ofoverexpression of FtsY on protein translocation were examined bypulse-chase experiments using hosts expressing heterologous proteins.

B. Licheniformis T399 was transformed with plasmid pLAT-IL3 containingthe human Interleukin-3 (h-IL-3) gene expressed from the B.licheniformis α-amylase promoter and provided with the B. licheniformisα-amylase signal sequence, or with the plasmid pLP10-AB containing theprochymosin gene under the same expression signals. The resultingstrains T399IL and T399Chy were transformed with plasmid pHBNFtsYcontaining the ftsY gene. As a control also both strains T399IL andT399Chy were transformed with the vector pHBNde.

Single colonies of all strains were inocculated in 5 ml of mediumstarvation medium s7+ (including Methionine and Cysteine) and grown at37° C. overnight. Aliquots of 200 μl were inocculated in 5 ml S7−(without Methionine and Cysteine) medium and grown for another 5 hours.

After growth to OD=0.3-0.7 a sample of 3.2 ml was centrifuged, washedwith S7− medium and resuspended into 3.2 ml fresh S7− medium. The samplewas incubated for 20 minutes at 37° C. and pulsed with 25μ (Ci L−[³⁵S]-Methionine (>1000 Ci/mmol) per ml during 60 seconds at 37° C. Thena chase was performed by addition of 50 μl (2 mg/ml) L-Methionine perml. The chase was stopped at different time points (0, 15, 30, and 60seconds) by mixing of 600 μl of the reaction with 600 μl ice cold 20%TCA, and incubation on ice for at least 30 minutes. The samples werecentrifugated, and the supernatant was used directly for immunoprecipitation, SDS-polyacrylamide gel electrophoresis andautoradiography using standard protocols.

The cell pellet was washed with 1 ml aceton, and dried. The cells wereresuspended in 50/l lysis buffer 10 mM Tris pH8, 25 mM MgCl, 200 mMNaCl, 5 mg/ml lysozyme) and incubated for 3 minutes at 37° C. Afteraddition of 50 μl TES (20 mM Tris, 2 mM EDTA, 2% SDS, pH8) the sampleswere boiled for 5 minutes. To the samples was added 900 μl of STDT (10mM Tris, 0.9% NaCl, 1% Triton, 0.5% Sodium deoxycholate, pHS.2), themixture was incubated for 15-60 minutes on ice, and the debris wasprecipitated. The supernatant was used for immuno precipitation withantiserum raised against h-IL-3 or Chymosin and subsequentSDS-polyacrylamide gel electrophoresis and autoradiography usingstandard protocols.

Overexpression of FtsY increased the secretion of the mature form ofinterleukin-3 and the processing of the precursor of prochymosin.

EXAMPLE 7 Effect of Overexpression of Ffh on the Secretion of HumanInterleukin-3

The ffh gene encoding the B. subtilis homologue of the eukaryotic SRP54protein was cloned as a PCR fragment obtained using primers based on thepublished DNA sequence (Honda et al, 1993). The gene was cloned into thevector pHBNde (see Example 1) under the control of the strongLactococcal P59 promoter to form plasmid pHBNFfh.

Pulse-chase experiments were performed as described in Example 6 with B.licheniformis strains containing both plasmid pHBNffh and plasmidpLAT-IL3 harbouring the human Interleukin-3 (h-IL-3) gene under theexpression and secretion signals of the B. licheniformis α-amylase gene.

As is shown in FIG. 9, the processing of h-IL-3 is very fast, as noprecursor can be detected. The amount of mature h-IL-3 in thesupernatant fraction is in all cases is higher in the samples obtainedfrom the strain overproducing Ffh compared to the strain containing onlythe vector plasmid.

EXAMPLE 8 Effects of Overexpression of scRNA on the Secretion of HumanInterleukin-3

The scr gene encoding the scRNA from B. subtilis was cloned as a PCRfragment using primers based on the published DNA sequence (Struck etal, 1989) and following the approach (including the same 5′ primer)described by Nakamura (Nakamura et al, 1992) introducing a HindIII sitejust upstream of the scr DNA and a SphI site downstream of theterminator sequence.

The P59 promoter was deleted from the vector pHB210 by replacement ofthe AlwNI-Smal fragment containing the origin of replication (ori)together with the P59 promoter by the AlwNI-PvuII fragment from vectorpBR322 containing only the same ori. This new vector pBHk was used toexchange the EcoRI-PvuII fragment for the EcoRI-BamRI (blunted by T4polymerase) containing the SPAC-penP-lacI cassette from pDG148 tocontract the vector pBHSpac.

The PCR fragment containing the scr gene was digested with HindIII andSphI and ligated into pBHSpac digested with the same restriction enzymesto construct plasmid pBHSscr. This way the sir gene was placed under thecontrol of the SPAC promoter.

Pulse-chase experiments were performed as described in Example 6 with B.licheniformis is strains containing both plasmid pBHSscr and plasmidpLAT-IL3 harbouring the human Interleukin-3 (h-IL-3) gene under theexpression and secretion signals of the B. licheniformis α-amylase gene.

Also in this case the processing of h-IL-3 was very fast, as noprecursor could be detected. However, the amount of mature h-IL-3 in thesupernatant fraction was in all cases higher in the samples obtainedfrom the strain overproducing scRNA compared to the strain containingonly the vector plasmid, or with plasmid pBHSscr in the absence of theinducer IPTG.

EXAMPLE 9 Effects of Simultaneously Overexpressed Signal RecognitionParticle components on the Secretion of Heterologous Proteins

The effects described in Examples 6, 7 and 8 were even more pronouncedwhen more than one of the components of the bacterial signal recognitionparticle were simultaneously expressed. For this purpose the ftsY genewas expressed from the IPTG inducible promoter located in the chromosomeas described in Example 4 by addition of 3 mM IPTG, while the componentsFfh or scRNA were expressed from their pHE210 derived vectors describedin Examples 7 and 8 above.

References

Ausubel et al. (1987), Current Protocols in Molecular Biology, JohnWiley and Sons Inc., New York.

Bernstein, H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, andP. Walter. 1989.

Nature (London) 340, 482-486.

Collier, D. N., 1994. J. Bacteriol. 176, 1937-1940.

Gilmore, R., 1993. Cell 75, 589-592.

Hann, B. C., M. A. Poritz, and P. Walter. 1989. J. Cell Biol. 109,3223-3230.

Hartl, F-U., S. Lecker, E. Schiebel, J. P. Hendrick, and W. Wickner.1990. Cell 63, 269-279.

Honda, K., K. Nakamura, M. Nishiguchi, and K. Yamane. 1993. J.Bacteriol. 175, 4885-4894.

Jeong, S. M., H. Yoshikawa, and H. Takahashi. 1993. Mol. Microbiol. 10,133-142.

Kim, Y. J., T. Rajapandi, and D. Oliver. 1994. Cell 87, 845-853.

Kumamoto, C. A., and J. Beckwith. 1983. J. Bacteriol. 154, 253-260.

Kumamoto, C. A., and J. Beckwith. 1985. J. Bacteriol. 163, 267-274.

Lill, R., W. Dowhan, and W. Wickner. 1990. Cell 60, 259-269.

Luirink, J., S. High, H. Wood, A. Giner, D. Tollerrey, and B.Dobberstein. 1992. Nature 359, 741-743.

Luirink, J., C. M. ten Hagen-Jongman, C. C. van der Weyden, B. Oudega,S. High, B. Dobberstein, and R. Kusters. 1994. EMBOJ. 13, 2289-2296.

Nakamura K., Y. Imai, A. Nakamura, and K. Yamane. 1992. J Bacteriol.174, 2185-2192.

Oliver, D. B., and J. Beckwith. 1981. Cell 25, 765-772.

Poritz, M. A., K. Strub, and P. Walter. 1988. EMBO J. 3, 3303-3310.

Ribes, U., K. R{circumflex over ( )}misch, A. Giner, B. Dobberstein, andD. Tollerrey. 1990. Cell 63, 591-600.

Romisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron, and B.Dobberstein. 1989. Nature 340, 478-482.

Sadaie, Y., H. Takamatsu, K. Nakamura, and K. Yamane. 1991. Gene 98,101-105.

Samuelsson, T. 1992. Nucleic Acids Res. 20, 5763-5770.

Schiebel, E., A. J. M. Driessen, F-U. Hartl, and W. Wickner. 1991. Cell64, 927-939.

Struck, J. C. R., R. K. Hartmann, H. Y . Toschka, and V. A. Erdmann.1989. Mol. Gen. Genet. 215, 478-482.

Struck, J. C. R., and V. A. Erdmann. 1990. Eur. J. Biochem. 192, 17-24.

Su, J-W., A. Boylan, S. M. Thomas, K. M. Dolan, D. B. Oliver, and C. W.Price. 1990. Mol. Microbiol. 4, 305-314.

Van der Vossen, J. M. B. M., D. van der Lelie, and G. Venema. 1987,Appl. Environ. Microb. 53, 2452-2457.

Van Dijl, J. M., A. de Jong, J. Vehmaanperd, G. Venema, and S. Bron.1992. EMBO J. 11, 2819-2828.

Yansura, D. G., and D. J. Henner. 1984. Proc.Natl.Acad.Sci. 81, 439-443.

What is claimed is:
 1. An isolated protein having the amino acidsequence of SEQ ID NO:8.
 2. An isolated polynucleotide encoding theamino acid sequence of SEQ ID NO:8.
 3. The isolated polynucleotide ofclaim 2 having the nucleic acid sequence of SEQ ID NO:
 7. 4. Anexpression vector comprising the polynucleotide of claim
 2. 5. Anexpression vector comprising the polynucleotide of claim
 3. 6. A hostcell comprising the expression vector of claim 4 or claim
 5. 7. The hostcell of claim 6 that is a Bacillus species.
 8. The host cell of claim 7,wherein said Bacillus species is selected from the group consisting ofBacillus subtilis, Bacillus amyloliquefaciens, Bacillus alcalophilus,Bacillus centus and Bacillus stearothenmophilus.
 9. The host cell ofclaim 7 further comprising a protein of interest.
 10. The host cell ofclaim 9 further a Bacillus Ffh protein.
 11. The host cell of claim 10further comprising a Bacillus scRNA.
 12. The host cell of claim 9,wherein the protein of interest is heterologous to the host cell. 13.The host cell of claim 9, wherein the protein of interest is homologousto the host cell.
 14. A transformed Bacillus host cell comprising (i) apolynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8and (ii) a DNA sequence encoding the heterologous protein.
 15. A methodfor producing a protein of interest comprising the steps of: a)culturing under suitable conditions a Bacillus host cell comprising aDNA encoding the protein of interest, said host cell transformed with aDNA encoding a protein having the amino acid sequence of SEQ ID NO:8 ora protein having an amino acid sequence at least 85% identical to SEQ IDNO:8. wherein said protein has a chaperone function; and b) allowingexpression of the protein of interest.
 16. The method of claim 15wherein the Bacillus of step a) further comprises a DNA sequenceencoding a Bacillus Ffh protein.
 17. The method of claim 16 wherein theBacillus further comprises a polynucleotide sequence encoding a BacillusscRNA.
 18. The method of claim 15, wherein said Bacillus is selectedfrom the group consisting of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus alcalophilus, Bacillus centus and Bacillusstearothermophilus.
 19. The method of claim 15, wherein the DNA encodingan amino acid sequence encodes the amino acid sequence of SEQ ID NO:8.20. A method for producing a heterologous protein from a gram-positivebacteria comprising the steps of: a) transforming a gram-positivebacterial host cell with a DNA molecule comprising i) a polynucleotidesequence encoding the amino acid sequence of SEQ ID NO:8 and ii) a DNAsequence encoding the heterologous protein, b) culturing the transformedbacterial host cell under suitable conditions, and c) allowingexpression and secretion of the heterologous protein.
 21. The method ofclaim 20, wherein the gram-positive bacteria is a species of Bacillus.22. The method of claim 21, wherein the Bacillus is selected from thegroup consisting of Bacillus subtilis, Bacillus amyloliquefaciens,Bacillus alcalophilus, Bacillus centus and Bacillus stearothermophilus.23. The method of claim 21 further comprising transforming said Bacillushost cell with a polynucleotide sequence encoding a Bacillus homologuefunctionally equivalent to the chaperone proteins selected from thegroup consisting of eukaryotic SRP54 protein and E. coli Ffh protein.24. The method of claim 21 further comprising transforming said Bacillushost cell with a polynucleotide sequence encoding a Bacillus subtilisFfh protein.
 25. A method for producing a protein of interest comprisingthe steps of: a) transforming a Bacillus host cell with a DNA moleculecomprising i) a polynucleotide sequence encoding the protein of interestand ii) a DNA encoding a protien having an amino acid sequence at least85% identical to the amino acid sequence of SEQ ID NO:8 and having achaperone function; b) culturing the transformed Bacillus host cellunder suitable conditions, and c) allowing expression and secretion ofthe protein of interest.